Potassium channels and human epileptic phenotypes:

Potassium channels and human epileptic phenotypes:

an updated overview

Chiara Villa^1*, Romina Combi¹

1University of Milano-Bicocca, Italy

Submitted to Journal:

Frontiers in Cellular Neuroscience ISSN:

1662-5102 Article type:

Review Article Received on:

28 Sep 2015 Accepted on:

15 Mar 2016

Provisional PDF published on:

15 Mar 2016

Frontiers website link:

www.frontiersin.org Citation:

Villa C and Combi R(2016) Potassium channels and human epileptic phenotypes: an updated overview.

Front. Cell. Neurosci. 10:81. doi:10.3389/fncel.2016.00081 Copyright statement:

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Provisional

1

Potassium channels and human epileptic phenotypes: an updated

1

overview

2 3 4

Chiara Villa

, Romina Combi

5

6

a

Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy 7

8 9 10 11 12 13 14 15 16

Words number: 5896 17

Running title: Potassium channels in epilepsy 18

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

*Correspondence:

41

Dr. Chiara Villa 42

University of Milano-Bicocca 43

Department of Surgery and Translational Medicine 44

Via Cadore, 48 45

20900 Monza (MB), Italy 46

chiara.villa@unimib.it 47

Provisional

2 ABSTRACT

48

Potassium (K

) channels are expressed in almost every cells and are ubiquitous in neuronal and 49

glial cell membranes. These channels have been implicated in different disorders, in particular in 50

epilepsy. K

channel diversity depends on the presence in the human genome of a large number of 51

genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K

52

channels were cloned and they represent the largest group of ion channels regulating the electrical 53

activity of cells in different tissues, including the brain. It is therefore not surprising that mutations 54

in these genes lead to K

channels dysfunctions linked to inherited epilepsy in humans and non- 55

human model animals.

56

This article reviews genetic and molecular progresses in exploring the pathogenesis of different 57

human epilepsies, with special emphasis on the role of K

channels in monogenic forms.

58 59 60 61 62

Keywords: K

channels, epilepsy, mutation, KCNT1, Kir channels, Kv channels 63

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

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3 INTRODUCTION

97

Epilepsy is one of the most common neurological disorders characterized by abnormal electrical 98

activity in the central nervous system (CNS) and recurrent seizures represent a cardinal clinical 99

manifestation. The phenotypic expression of each seizure is determined by the original point of the 100

hyperexcitability and its degree of spread in the brain (Steinlein, 2004). Several brain defects due to 101

membrane instability could cause epilepsy.

102

In the last two decades, gene defects underlying different forms of epilepsy have been identified and 103

most of these genes code for ion channels, which thus appear as important players in the 104

etiopathogenesis of idiopathic epilepsy. Indeed, several epileptic phenotypes have been associated 105

to dysfunctions of potassium (K

) channels (Brenner and Wilcox, 2012). It has been recently 106

proposed to name such epilepsies as “K

channelepsies” (D’Adamo et al., 2013). These channels 107

play a major role in neuronal excitability and their importance is related to the level of their 108

expression in subcellular domain, individual cell, or circuit (Cooper, 2012). K

channels are also 109

involved in setting the inward-negative resting membrane potential. Based on their structures, 110

biophysical characteristics, pharmacological sensitivities and physiology, these channels are 111

classified as voltage-gated (Kv), inwardly rectifying (Kir), sodium (Na)-activated channels or Ca

- 112

activated channels (Table 1) (González et al., 2012).

113

Herein we report an updated discussion on the role of mutations in K

channels (Table 2) in the 114

pathogenesis of human epilepsy.

115 116 117

VOLTAGE-GATED K

CHANNELS (Kv) 118

The Kv channels are widely expressed both in the central and in peripheral nervous system where 119

they are involved in several processes (e.g., the regulation of the duration of action potentials, the 120

modulation of the neurotransmitter release, the control of the electrical properties and the firing of 121

neurons). Kv channels generally regulate outward K

currents that contribute to membrane 122

repolarization and hyperpolarization, thus limiting the neuronal excitability. Moreover, they 123

actively participate in cellular and molecular signaling pathways that regulate the life and death of 124

neurons, such as apoptosis, channel phosphorylation or cell proliferation (Shah and Aizenman, 125

2014). In particular, neuronal cell apoptosis is correlated to an increased expression of Kv channels 126

at the plasma membrane, thus facilitating more K

efflux and a loss of cytosolic K

. This drop in the 127

intracellular K

concentration actives pro-apoptotic enzymes, such as nuclease or caspase that can 128

trigger downstream apoptotic signals culminating in DNA fragmentation or degradation (Leung et 129

al., 2010).

130

In human genome, forty different genes encoding for Kv channels were reported and subdivided 131

into twelve sub-families (Kv1 through Kv12) (Gutman et al., 2005). Mammalian Kv channels are 132

tetramers, composed of α-subunits that line an ion pore. Each α-subunit shows six α-helical 133

transmembrane domains (S1–S6), a membrane-reentering P loop between S5 and S6, and cytosolic 134

N- and C-termini. The S5-P-S6 segments constitute the ion conduction pore, while the S1–S4 135

sequences are critical for the voltage-sensing and gating of the channel (Brenner and Wilcox, 2012).

136

Furthermore, α-subunits can bind to regulatory β subunits (Kvβ1, Kvβ2 and Kvβ3) as well as to 137

other Kv channel-interacting proteins. This variability in the channel interactions results in strong 138

modifications of the channel properties (McKeown et al., 2008).

139

The following Kv subfamilies have been associated with either epilepsy or other disorders showing 140

seizures.

141 142

Kv1 143

The Kv1 subfamily plays an essential role in the initiation and shaping of action potentials. These 144

channels are expressed at the soma, axons, synaptic terminals, and proximal dendrites. The most 145

abundant Kv1 α-subunits are Kv1.1, Kv1.2, and Kv1.4. These subunits are differentially expressed 146

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4 and their composition is dependent upon the brain region, cell type and subcellular localization 147

(Robbins and Tempel, 2012).

148

Heterozygous mutations in the KCNA1 gene, encoding the Kv1.1 α subunit, were associated with 149

episodic ataxia type 1 (EA1), a dominantly inherited disorder characterized by generalized ataxia 150

attacks and spontaneous muscle quivering (Browne et al., 1994). Interestingly, a subset of patients 151

with familial EA1 shows epileptic seizures, suggesting that Kv1.1 dysfunctions may play a role in 152

the pathophysiology of epilepsy (Spauschus et al., 1999; Zuberi et al., 1999; Eunson et al., 2000).

153

Loss-of function mutations reported in the KCNA1 gene of EA1 patients cause reduced current 154

amplitude thus contributing to seizures susceptibility (Adelman et al., 1995; Browne et al., 1994;

155

D’Adamo et al., 1999; Imbrici et al., 2006).

156

In support of the hypothesis of an epileptogenic role of KCNA1 mutations, several knock-out mouse 157

models for this gene developed an epileptic phenotype (Smart et al., 1998; Rho et al., 1999).

158

Biochemical and biophysical studies demonstrated a colocalization of Kv1.1 and Kv1.2 subunits in 159

several subcellular brain regions and that they could form heteromeric channels, which are reported 160

as profoundly altered by EA1 mutations (D’Adamo et al., 1999).

161

Notably, a Kv1.2 knock-out mouse model displayed increased seizure susceptibility (Brew et al., 162

2007). In this regard, Syrbe and collaborators recently identified de novo loss or gain-of-function 163

mutations in KCNA2 gene (Table 2), encoding the Kv1.2 channel, in patients showing mild to 164

severe epileptic encephalopathy (Syrbe et al., 2015). A role of Kv1.2 was also suggested by another 165

case report describing a de novo mutation, leading to the p.Arg297Gln amino acid substitution in a 166

patient affected by ataxia and myoclonic epilepsy (Pena and Coimbra, 2015).

167 168

Kv4 169

The Kv4 channels are highly expressed in the brain and mediate the main dendritic A-currents 170

which critically regulate action potential back-propagation and the induction of specific forms of 171

synaptic plasticity. In particular, the Kv4.2 subunit is a key component of the A-type potassium 172

current in the CNS (I

) (Birnbaum et al., 2004).

173

In 2006, Singh and collaborators described a truncation mutation (p.Asn587fsX1) in the Kv4.2 174

channel encoded by the KCND2 gene, in a patient affected by temporal lobe epilepsy (TLE). This 175

mutation causes a frame-shift, leading to a premature termination codon and consequently to a 176

Kv4.2 channel haploinsufficiency (Singh et al., 2006). Recently, a whole exome sequencing study 177

identified a de novo gain-of-function mutation (p.Val404Met) in KCND2. The mutation was found 178

in monozygotic twins affected by autism and severe intractable seizures and occurred at a highly 179

conserved residue within the C-terminus of the S6 transmembrane region of the ion pore. A 180

functional analysis of mutated channels revealed a significantly slowed channel inactivation (Lee et 181

al., 2014).

182

Very recently, an involvement of Kv4.3 subunits in epilepsy was also suggested by the 183

identification of a de novo mutation (p.Arg293_Phe295dup) in the relevant KCND3 gene causing a 184

severe channel dysfunction in a patient with complex early onset cerebellar ataxia, intellectual 185

disability, oral apraxia and epilepsy. This mutation results in the duplication of a RVF (Arginine- 186

Valine-Phenylalanine) motif in the S4 segment and leads to a more positively charged voltage- 187

sensor domain, altering the voltage-dependent gating properties of the channel. In details, the 188

p.Arg293_Phe295dup mutation induced a strong depolarizing shift in the voltage dependence of 189

both the activation (about +59.3 mV) and inactivation ( +62 mV) of the channel (Smets et al., 190

2015).

191 192

Kv7 193

KCNQ (Kv7) channels are low-threshold activated voltage-gated potassium channels. Among the 194

five known isoforms, KCNQ2–5 are expressed throughout the nervous system, whereas KCNQ1 is 195

mostly expressed in cardiac tissue. The KCNQ2 gene is the most commonly reported as mutated in 196

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5 epilepsy. Its mutations cause neonatal epilepsies with wide phenotypic heterogeneity, ranging from 197

benign familial neonatal seizures (BFNS) with normal cognition and unremarkable neuroimaging to 198

early onset epileptic encephalopathies (EOEEs) with mental retardation, suppression-burst 199

electroencephalography (EEG) and distinct neuroradiologic features (Singh et al., 1998;

200

Weckhuysen et al., 2012; Soldovieri et al., 2014). More than 80 different mutations in KCNQ2, 201

consisting of missense, non-sense, truncations, splice site defects and frame-shift mutations, as well 202

as sub-microscopic deletions or duplications, were described and most of them are found in the pore 203

region and the large intracellular C-terminal domain (Lee et al., 2009). Functional studies suggested 204

a strict phenotype/genotype correlation between disease severity and functional properties of mutant 205

channels (Miceli et al., 2013). KCNQ2 is a primary player that mediates neuronal muscarinic (M) 206

currents: the opening of this channel or of heterogeneous KCNQ2/KCNQ3 complexes inhibits 207

initiation of action potential and thus suppresses neuronal excitability (Brown and Passmore, 2009).

208

Mutations in KCNQ3 gene have been described in families affected with benign epilepsy with 209

variable age of onset and good outcome (Zara et al., 2013; Griton et al., 2015) or in a patient with 210

benign childhood epilepsy with centrotemporal spikes (BECTS) (Fusco et al., 2015). However, two 211

recent reports suggested that mutations in KCNQ3, similarly to KCNQ2, can be also found in 212

patients with more severe phenotypes, including intellectual disability. In particular, they described 213

KCNQ3 mutations in patients with early-onset epilepsy and neurocognitive deficits (Soldovieri et 214

al., 2014; Miceli et al., 2015; Table 2).

215

Mutations in the KCNQ1 gene were associated with a particular form of long QT syndrome, the 216

LQT1 (Wang et al., 1996). Interestingly, some authors observed that epilepsy occurred in mouse 217

lines bearing dominant human LQT1 mutations in this channel, which caused syncope and sudden 218

death (Goldman et al., 2009). Moreover, genetic variants in the KCNQ1 gene were reported in three 219

cases of sudden unexpected death in epilepsy (SUDEP), a catastrophic complication of human 220

idiopathic epilepsy with unknown causes. However, the relationship of these variants to the disease 221

remains to be elucidated (Yang et al., 2009; Partemi et al., 2015). The evidence that KCNQ1 genetic 222

variations may confer susceptibility for recurrent seizure activity increasing the risk of sudden death 223

is further supported by the description of a pathogenic KCNQ1 variant (p.Leu273Phe) in a family 224

featuring LQTS and epilepsy (Tiron et al., 2015).

225 226

Kv8 227

The KCNV2 gene encodes the voltage-gated K

channel Kv8.2. This subunit is 228

electrophysiologically silent when assembled in homotetramer. Otherwise, it significantly reduces 229

the surface expression of the resulting channels and influences their biophysical properties when 230

involved in the formation of functional heterotetramers with Kv2 subunits (Czirják et al., 2007).

231

Kv2.1 and Kv8.2 show significant regional overlap: within the hippocampus, transcripts for both 232

KCNV2 and KCNB1, which encodes Kv2.1, are detected in excitatory neurons of the pyramidal cell 233

layers and the dentate gyrus. Similarly, both of them are abundantly expressed in the cortex 234

(Maletic-Savatic et al., 1995). Their regional colocalization is consistent with an effect of Kv8.2 235

variants on Kv2.1 channels within cells critically important for seizure generation and propagation.

236

A support of the involvement of KCNV2 in seizure pathogenesis was provided by the identification 237

of non-synonymous variants in two unrelated children showing epilepsy: p.Arg7Lys and 238

p.Met285Arg. In particular, the p.Arg7Lys was found in a patient affected by febrile and afebrile 239

partial seizures, whereas the p.Met285Arg was reported in a case of epileptic encephalopathy and 240

severe refractory epilepsy. The functional characterization of these variants demonstrated that they 241

both enhanced Kv8.2-mediated suppression of Kv2.1 currents, suggesting a role in decreasing 242

delayed rectifier K

current in neurons, therefore increasing cells excitability. Moreover, the 243

p.Met285Arg caused a shift in the voltage-dependence of activation as well as slower activation 244

kinetics, in accordance with the more severe clinical phenotype of the patient (Jorge et al., 2011).

245 246 247

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6 Kv11-HERG

248

The human ether-a-go-go-related gene (hERG, also known as KCNH2) encodes the pore-forming 249

subunit of the rapid component of the delayed rectifier K

channels, Kv11.1, which are expressed in 250

several tissues, mostly in brain and heart. In the brain, Kv11.1 channels regulate neuronal firing and 251

modulate the excitability of GABAergic and dopaminergic neurons. The same channel exerts a 252

different function in the heart being involved in the regulation of membrane potentials in the 253

ventricles (Vandenberg et al., 2012).

254

Mutations in the KCNH2 gene were reported to cause type 2 long QT syndrome (LQT2), a rare 255

inherited ion channel disorder characterized by prolonged QT interval and predisposing patients to 256

ventricular arrhythmias that can lead to syncope and sudden cardiac death (SCD). LQT2 syndrome 257

is frequently misdiagnosed as epilepsy due to seizures that are triggered by cerebral hypoperfusion 258

during a ventricular arrhythmia, therefore suggesting a possible link between epilepsy and cardiac 259

arrhythmias, as described by several clinical reports (Johnson et al., 2009; Keller et al., 2009;

260

Omichi et al., 2010; Tu et al., 2011; Zamorano-León et al., 2012; Partemi et al., 2013). In particular, 261

a seizure phenotype was reported in about 30% of unrelated LQTS patients carrying pathogenic 262

variants in the KCNH2 gene, suggesting that mutations in the Kv11.1 channel associated with 263

LQTS may also predispose to seizure activity (Johnson et al., 2009). Moreover, a post-mortem 264

study identified nearly 13% of LQTS pathogenic variants in the KCNH2 and SCN5A genes in 265

epileptic samples. In particular, regarding KCNH2, two non-synonymous mutations have been 266

identified: p.Arg176Trp and p.Arg1047Leu (Tu et al., 2011). Another study on three families 267

showing a history of seizures and LQTS2 lead to the identification of three novel KCNH2 268

mutations: p.Tyr493Phe, Ala429Pro and Thr74ArgfsTer32 (also named p.del234-241). In vitro 269

functional analyses of all these variants showed a loss of hERG potassium channel function with a 270

reduction of the current, suggesting a dominant negative effect (Keller et al., 2009). Omichi and 271

collaborators reported a case of a man with long history of epilepsy and referred for cardiologic 272

evaluation, showing the p.Arg534Cys mutation (Omichi et al., 2010). In addition, other authors 273

identified a nonsense mutation (p.Arg863X) leading to a 296-amino acid deletion (Zamorano-León 274

et al., 2012) while a loss-of-function mutation (p.Ile82Thr) was reported in a pedigree featuring 275

LQTS, idiopathic epilepsy and increased risk of sudden death (Partemi et al., 2013).

276 277

AUXILIARY SUBUNITS OF Kv CHANNELS 278

Kv channel functional diversity is enhanced by coassembly with a wide array of auxiliary subunits, 279

which cannot form functional channels alone but which can greatly impact channel function upon 280

coassembly with α subunits to form hetero-oligomeric complexes (Trimmer, 1998). Defects in these 281

subunits may affect Kv channel function and network excitability, resulting thus in an increase of 282

seizure susceptibility. Several subunits have been identified, including β-subunit (Kvβ), leucine-rich 283

glioma-inactivated-1 (Kv

) and K

channel-interacting protein (Kv

).

284 285

Kvβ 286

Kvβ subunits are cytoplasmatic proteins critical for the correct membrane localization and normal 287

biophysical properties of voltage-gated K

channels. Variations in the expression of different Kvβ 288

genes and their isoforms could significantly impact K

channel function, especially with respect to 289

inactivation kinetics. In the mammalian genome three genes encode Kvβ subunits: Kvβ1, Kvβ2 and 290

Kvβ3 (Pongs and Schwarz, 2010). Interestingly, Kvβ2 knockout mouse models were characterized 291

by cold-swim induced tremors and occasional seizures, suggesting thus a role of this subunit in the 292

regulation of neuronal excitability (McCormack et al., 2002). An association between the severity 293

of seizures and the loss-of-function of the KCNAB2 gene that encodes the β2 subunit was reported 294

(Heilstedt et al., 2001). In particular, the hemizygous deletion of KCNAB2 identified in this 295

manuscript in epileptic patients suggested that haploinsufficiency of this gene may represent a 296

significant risk factor for epilepsy: the lack of the β subunit would reduce K

channel-mediate 297

membrane repolarization and increase neuronal excitability (Heilstedt et al., 2001).

298

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7 Kv

299

The leucine-rich glioma-inactivated-1 (LGI1) is the best characterized LGI family protein, highly 300

expressed in neurons, which encodes a secreted protein containing two domains (a leucine-rich 301

repeat domain (LRR) and a β-propeller domain called EPTP) that mediate protein-protein 302

interactions. LGI1 binds to the presynaptic voltage-gated potassium channel Kv1.1 and prevents Kv 303

channel inactivation mediated by the β subunit of the channel (Schulte et al., 2006). The LG1 gene 304

was find to be mutated in approximately 50% of ADLTE (autosomal dominant lateral temporal lobe 305

epilepsy) families: more than 30 disease-causing mutations in LGI1 gene have been associated so 306

far with this focal epilepsy that is characterized by good response to antiepileptic drugs and with a 307

juvenile onset (Kalachikov et al., 2002; Morante-Redolat et al., 2002; Dazzo et al., 2015). In 308

particular, almost all mutations are missense, splice-site or short indels (Ho et al., 2012; Nobile et 309

al., 2009) while only a single microdeletion has been reported (Fanciulli et al., 2012). Certain LGI1 310

mutants (typically non-secreted mutants) fail to prevent channel inactivation resulting in more 311

rapidly closing channels, which extends presynaptic depolarization and leads to increased calcium 312

(Ca

) influx. Consequently, release of neurotransmitter is increased excessively and may induce 313

focal seizures (Nobile et al., 2009). Moreover, it was demonstrated that the loss of LGI1 gene in 314

mice induced lethal epilepsy, suggesting its essential role as an antiepileptogenic ligand. LGI1 may 315

serve as a major determinant of brain excitation and the LGI1 gene-targeted mouse could provide a 316

good model for human epilepsy (Fukata et al., 2010).

317 318

Kv

319

The K

channel-interacting proteins (KChIPs 1–4) compose a subfamily of neuronal Ca

sensor 320

proteins that modulate trafficking, targeting to the plasma membrane, as well as turnover and 321

endocytosis of Kv4 channels (An et al., 2000). Among KChIPs, KChIP2 is abundantly expressed in 322

hippocampal pyramidal cells and represents the major target of Kv4 α subunits to form a complex 323

essential for I

regulation in hippocampal neurons (Rhodes et al., 2004). This current has been 324

found to be reduced in the presence of a deletion in the KChIP2 gene by Wang and collaborators.

325

The authors thus suggested that it may increase susceptibility to seizures (Wang et al., 2013).

326

Moreover, they also hypothesized a role of KChIP2 in SUDEP risk (Wang et al., 2013), since 327

KChIP2 knockout mice were previously shown to be highly susceptible to induced arrhythmias 328

(Kuo et al., 2001). In conclusion, these data suggested that loss-of-function mutations in modulatory 329

subunits could increase the susceptibility to seizures and cardiac arrhythmias, thereby providing a 330

unified mechanism for a neurocardiac syndrome such as SUDEP.

331 332 333

INWARDLY RECTIFYING POTASSIUM CHANNELS 334

Inwardly rectifying K

(Kir) channels are widely expressed in several excitable and non-excitable 335

tissues playing a key role in the maintenance of the resting membrane potential and consequently in 336

the regulation of cell excitability. Approximately 15 Kir clones forming either homotetramers or 337

heterotetramers were identified and grouped in 7 different families based on sequence similarity and 338

functional properties: Kir1-Kir7 (Hibino et al., 2010). Generally, Kir channels showed the greater 339

conductance at negative potentials in respect to the equilibrium potential for K

(E

), while an 340

inhibition of the outward flow of K

ions caused by both Mg

and polyamines was reported at 341

more positive values (Lopatin et al, 1994). Several Kir channels have been associated with epileptic 342

phenotypes and, in particular, Kir2.1, Kir3, Kir4 and Kir6.

343 344

Kir2.1 345

The Kir2.1 channel is encoded by the KCNJ2 gene whose expression is reported in several brain 346

areas (Karschin et al., 1996) as well as in astrocytes where they control astrocyte-mediated K

347

buffering in combination with Kir4.1 (Jabs et al., 2008; Chever et al., 2010).

348

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8 Several mutations impairing the channel functionality were reported in the KCNJ2 of Andersen- 349

Tawil syndrome (ATS) patients (Haruna et al., 2007; Chan et al., 2010; Guglielmi et al., 2015; see 350

Table 2 for mutation details). On the other hand, Kir2.1 gain-of-function mutations cause the type-3 351

variant of the short QT syndrome (SQT3s) which results in QT shortening and increased risk of 352

sudden cardiac death (Priori et al., 2005). Recently, some authors detected a novel mutation 353

(p.Lys346Thr) in the KCNJ2 in monozygotic twins displaying SQT3s and autism-epilepsy 354

phenotype, suggesting the existence of a Kir2.1 role in neuropsychiatric disorders and epilepsy.

355

Functional studies revealed that this mutation causes an increase of the channel’s surface expression 356

and stability at the plasma membrane, a reduction in protein degradation and an altered protein 357

compartmentalization (Ambrosini et al., 2014).

358 359

Kir3-GIRK 360

The G-protein-coupled Kir (GIRK) channels belong to the subfamily of Kir3 that are important 361

regulators of electrical excitability in both cardiomyocytes and neurons (Slesinger et al., 1995).

362

Different types of neurotransmitters, such as acetylcholine, dopamine, opioids, serotonin, 363

somatostatin, adenosine and GABA, activate these channels by stimulating their G-protein coupled 364

receptors (GPCRs). This results in a final membrane hyperpolarization and inhibition of cell 365

excitability due to the activation of an outward flux of K

ions (Krapivinsky et al., 1995; Slesinger 366

et al., 1995). Mammals express four GIRK channel subunits (GIRK1-4, also named Kir3.1-3.4), 367

encoded by KCNJ3, KCNJ6, KCNJ9 and KCNJ5, respectively. These four subunits can form homo 368

or heterotetramers with unique biophysical properties, regulation and distribution (Lüscher and 369

Slesinger, 2010).

370

Alterations in GIRK channel function have been associated with pathophysiology of severe brain 371

disorders, including epilepsy. In this regard, a GIRK2 knockout mouse model resulted to be more 372

susceptible to develop both spontaneous or induced seizures in respect to wild type mice (Signorini 373

et al., 1997). In particular, mice carrying a p.Gly156Ser mutation displayed an epileptic phenotype 374

(Patil et al., 1995). Indeed, this mutation has been found to alter the putative ion-permeable, pore- 375

forming domain of the channel, inducing Ca

overload in cells and reducing channel availability, 376

leading thus to neurodegeneration and seizures susceptibility (Slesinger et al., 1996).

377

An increased expression of GIRK channels was observed in rat brain after an electroconvulsive 378

shock, probably altering the excitability of granule cells and the functions of neurotransmitter 379

receptors which are coupled to these channels (Pei et al., 1999). Another evidence in support of a 380

role of GIRK channels in epilepsy was provided by the demonstration that ML297, a potent and 381

selective activator of GIRK channels, showed epileptogenic properties in mice (Kaufmann et al., 382

2013). On the other hand, the inhibition of GIRK channel activity by drugs causes seizures 383

(Mazarati et al., 2006). All these considerations imply that changes in Kir3 channel activity may 384

alter the susceptibility to seizures.

385 386

Kir4 387

Among Kir4 channels, the Kir4.1, encoded by the KCNJ10 gene, is the only one that has been 388

associated to epilepsy. This subunit can assemble itself in homomeric channels or it can constitute 389

heterotetramers in combination with Kir5.1 (KCNJ16) (Pessia et al., 2001). Kir4.1 expression has 390

been detected primarily in the thalamus, cortex, brainstem and hippocampus (Higashi et al., 2001).

391

Kir4.1 channels play a key role in maintaining resting membrane potential by transporting K

from 392

the extracellular space into glial cells in the CNS (Nishida and MacKinnor, 2002).

393

Alterations of Kir4.1 channels have been linked to seizure susceptibility in both mice (Ferraro et al., 394

2004) and humans (Buono et al., 2004). Conditional Kir4.1 knockout mice in astrocytes have been 395

found to display premature lethality and severe seizures prior to death (Djukic et al., 2007), 396

supporting the idea of a pathophysiological relationship of the Kir4.1 impairment with epilepsy.

397

Concerning human Kir4.1, a linkage study identified a missense variation (p.Arg271Cys) as 398

Provisional

9 associated with epileptic phenotypes (Buono et al., 2004). However, the variant did not result to 399

have functional effects in vitro (Shang et al., 2005). Mutations in this gene were also reported in 400

EAST syndrome (also named SeSAME) patients, a rare condition showing epileptic seizures among 401

other signs (Bockenhauer et al., 2009; Scholl et al., 2009; Freudenthal et al., 2011; see Table 2 for 402

mutation details).

403

Single nucleotide variations in Kir4.1 were detected in the DNA of TLE patients presenting with 404

hippocampal sclerosis and antecedent febrile seizures, supporting the importance of KCNJ10 as a 405

candidate gene for seizures susceptibility (Heuser et al., 2010).

406

Interestingly, several authors reported a strong association between epilepsy and autism spectrum 407

disorders (ASDs) and an “autism-epilepsy phenotype” has been proposed (Tuchman et al., 2005, 408

Lee et al., 2015). Indeed, a mutational screening of KCNJ10 in 52 children affected by cryptogenic 409

epilepsy identified two heterozygous mutations (p.Arg18Gln and p.Val84Met) in three children of 410

two unrelated families displaying seizures, ASDs and intellectual disability. The functional 411

consequences of these mutations appeared to be a gain-of-function mechanism. These findings 412

suggest that an abnormal K

homeostasis in the brain may increase the susceptibility to this 413

“autism-epilepsy phenotype” (Sicca et al., 2011). A common mechanism between autism and 414

epilepsy could be the impairment of astrocytic-dependent K

buffering, altering neuronal 415

excitability and synaptic function.

416 417

Kir6-K

418

The adenosine triphosphate (ATP)-sensitive K

(K

) channels are widely distributed in various 419

tissues where they couple cell metabolism to cell excitability. These channels are assembled by an 420

inward rectifier K

channel pore (Kir6.1/Kir6.2) and an ATP-binding regulatory subunit, named 421

sulfonylurea receptor (SUR1/SUR2A/SUR2B) (Olson and Terzic, 2010). Neuronal K

channels 422

are mainly constituted by a coassembly of Kir6.2/SUR1 subunits. (Inagaki et al., 1995).

423

Several gain-of-function mutations were detected in the Kir6.2 (KCNJ11) or the SUR1 subunit 424

(ABCC8). These mutations are responsible for developmental delay, epilepsy and neonatal diabetes 425

(DEND), accounting for approximately 40% of cases and caused a decrease in the ability of ATP to 426

block the K

channel. This results in more fully openings of the channel at physiologically 427

relevant concentrations of ATP, thus increasing the K

current (Hattersley and Ashcroft, 2005).

428

Nevertheless, the pathophysiological mechanism leading to epilepsy remains to be elucidated.

429

Probably, elevated levels of extracellular glucose and intracellular ATP attenuate K

channels, 430

producing a more excitable state (Huang et al., 2007). Moreover, mice lacking Kir6.2 are vulnerable 431

to hypoxia, exhibiting a reduced threshold for generalized seizure (Yamada et al., 2001). Transgenic 432

mice, overexpressing the SUR1 gene in the forebrain, show a significant increase in the threshold 433

for kainate-induced seizures (Hernandez-Sanchez et al., 2001).

434 435

SODIUM-ACTIVATED POTASSIUM CHANNELS (K

) 436

The Na

-activated K

channels (K

) are found in neurons throughout the brain and are responsible 437

for delayed outward currents named I

. These currents regulate neuronal excitability and the rate 438

of adaption in response to repeated stimulation at high frequencies. In many cases, I

is mediated 439

by the phylogenetically related K

channel subunits Slack and Slick (Bhattacharjee and 440

Kaczmarek, 2005). Like the Kv channels, these subunits have six hydrophobic, transmembrane 441

segments (S1–S6) with a pore P-domain between S5 and S6 and a large cytoplasmatic C-terminal 442

domain containing two regulators of K

conductance (RCK) domains that are likely to be sites for 443

Na

-binding and channel gating. The Slack subunit binds with Slick to form heterotetrameric 444

channel complexes (Kaczmarek, 2013). Slack has been associated with different epilepsy 445

phenotypes.

446 447 448

Provisional

10 SLACK

449

The KCNT1 gene encodes the K

channel subunit KCNT1, called Slack (sequence like a calcium- 450

activated potassium channel, also known as K

or Slo2.2). KCNT1 is highly expressed in the 451

brain but also in the heart and the kidney at lower levels. Concerning brain, it is not widely 452

expressed in the cortex but it is found in neurons of the frontal cortex (Bhattacharjee et al., 2002), 453

consistent with its known role in the pathogenesis of autosomal dominant nocturnal frontal lobe 454

epilepsy (ADNFLE) (Heron et al., 2012). While KCNT1 channels are thought to play important 455

roles in modulating the firing patterns and general excitability of many types of neurons, their 456

precise function is yet to be resolved.

457

Mutations in KCNT1 gene have been found in different epilepsy syndromes: ADNFLE (Heron et 458

al., 2012; Kim et al., 2014; Møller et al., 2015), epilepsy of infancy with migrating focal seizures 459

(EIMFS, previously known as malignant migrating partial seizures in infancy, MMPSI or also more 460

recently as malignant migrating focal seizures of infancy, MMFSI) (Barcia et al., 2012; Ishii et al., 461

2013; Ohba et al., 2015; Rizzo et al., 2016) and other types of EOEEs, (Vanderver et al., 2014;

462

Ohba et al., 2015), including Ohtahara syndrome (OS) (Martin et al., 2014). The involvement of 463

KCNT1 in these distinct disorders suggests that KCNT1 mutations may cause a spectrum of focal 464

epilepsies (Møller et al., 2015). Patients displaying KCNT1 mutations have a very high occurrence 465

of severe mental and intellectual disability.

466

Four missense mutations (p.Arg398Gln, p.Tyr796His, p.Met896Ile and p.Arg928Cys) in KCNT1 467

gene were reported to be associated with ADNFLE cases showing comorbidities of intellectual 468

disability and psychiatric features (Heron et al., 2012). This is in contrast to ADNFLE patients 469

without mutations in KCNT1 gene, where intelligence and other neurologic functions are largely 470

unimpaired (Philips et al., 1998). Mutations are clustered around the RCK and cytoplasmatic NAD

471

binding domain (Heron et al., 2012), the site that regulates the channel sensitivity to Na

472

intracellular concentrations (Tamsett et al., 2009). A complete penetrance is reported in ADNFLE 473

families showing KCNT1 mutations (Heron et al., 2012) with the exception of a non-penetrant case 474

(Møller et al., 2015).

475

Interestingly, Møller et al. reported that a KCNT1 mutation (p.Arg398Gln) can lead to either 476

ADNFLE or EIMFS within the same family, indicating that genotype-phenotype correlations are 477

not straightforward (Møller et al., 2015). Similarly, a more recent study showed that the 478

p.Gly288Ser mutation could cause both phenotypes, probably due to genetic modifiers or 479

environmental factors (Kim et al., 2014). Nevertheless, this association was unexpected since in 480

vitro studies demonstrated that mutations associated with MMFSI caused a significantly larger 481

increase in current amplitude than those associated with ADNFLE (Milligan et al., 2014).

482

Concerning EIMFS, in addition to the above mentioned p.Gly288Ser and p.Arg398Gln, several 483

additional mutations have been identified, including p.Val271Phe, p.Arg428Gln, p.Arg474Gln, 484

p.Met516Val, p.Lys629Asn, p.Ile760Met, p.Pro924Leu and p.Ala934Thr (Barcia et al., 2012; Ishii 485

et al., 2013; Mikati et al., 2015; Ohba et al., 2015; Rizzo et al., 2016). These are clustered not only 486

around the RCK and NAD

binding domain of the protein, but also within its S5 transmembrane 487

segment, indicating that the alteration of other regions of KCNT1 could also be pathogenic (Ishii et 488

al., 2013; McTague et al., 2013; Kim et al., 2014) 489

Finally, two KCNT1 mutations were associated with other forms of EOEEs, strengthening once 490

again the existence of a wide phenotypic spectrum of KCNT1 mutations. In particular, the 491

p.Phe932Ile was detected in a patient affected by EOEEs whereas the p.Ala966Thr was found in 492

one showing OS. Both of them are clustered around the RCK and NAD

binding domains of the 493

protein (Martin et al., 2014; Vanderver et al., 2014; Ohba et al., 2015).

494

The effect of nine different mutations in KCNT1 gene that give rise to these distinct forms of 495

epilepsy was examined and it was demonstrated that they all result in channels displaying a strong 496

gain-of-function phenotype: all of them produced many-fold increases in current amplitude as 497

compared with the wild-type channel. This could greatly increase the cooperativity in channel 498

gating that is detected in clusters of multiple channels (Kim et al., 2014).

499

Provisional

11 CALCIUM-ACTIVATED POTASSIUM CHANNELS (K

)

500

Ca

-activated K

channels are highly conserved complexes thought to play a critical role in 501

neuronal firing properties and circuit excitability in the human brain. Three groups of Ca

-activated 502

K

channels can be distinguished: large conductance (BK

), intermediate conductance (IK

), and 503

small conductance (SK

) channels (N’Gouemo, 2011). The opening of these channels is in 504

response to an increase in Ca

concentration and a depolarization of the membrane potential, which 505

in turn causes a secondary hyperpolarization reestablishing the membrane potential as well as Ca

506

levels. Otherwise it can produce an afterhyperpolarization to potentials more negative than the 507

resting membrane potential (Latorre and Brauchi, 2006; Nardi and Olesen, 2008). To date, only the 508

association between K

channel and epilepsy has been demonstrated.

509 510

K

511

KCNMA1 gene encoded the α-subunit of the large conductance K

channels. They show the 512

typical tetrameric structure of K

channels, with four α-subunits each displaying seven 513

transmembrane segments, with a unique S0 segment, and the charged S4 segment conferring the 514

voltage-dependence. Ca

sensitivity comes instead from the bulky C-terminal tail that includes a 515

negatively charged, high-affinity Ca

binding region (Jiang et al., 2001) and the double negative 516

charged RCK-domain. These channels could associate with four different types of β subunits (β1- 517

β4, each encoded by a specific gene KCNMB1-4) which modulated channel function uniquely (Orio 518

et al., 2002).

519

K

channels play a role in promoting high neuronal frequency firing which is consistent with 520

their predominant expression in axon and presynaptic terminals of neurons located in brain regions 521

(e.g. hippocampus and cortex) frequently involved in epilepsy (Gu et al., 2007; Martire et al., 2010).

522

The involvement of these channels in epilepsy was suggested not only by their localization but also 523

by studies on animal models. In this regard, it has been demonstrated in mice highly susceptible to 524

convulsions that the inhibition of K

channels is sufficient to block cortical bursting activity (Jin 525

et al., 2000). Moreover, the loss of β4 subunits in K

knockout mice promoted the excitatory 526

synaptic transmission, resulting in temporal cortex seizures (Brenner et al., 2005). Finally, 527

Ermolinsky and collaborators demonstrated a deficit of KCNMA1 expression in the dentate gyrus in 528

animal models, hypothesizing therefore its critical role in the pathogenesis of mesial temporal lobe 529

epilepsy (mTLE) (Ermolinsky et al., 2008).

530

An association between K

channels and epilepsy has also been observed in humans. A missense 531

mutation in KCNMA1 (p.Asp434Gly) was detected in a large family with generalized epilepsy and 532

paroxysmal dyskinesia. Functional studies revealed an increased Ca

sensitivity predicting a gain- 533

of-function and neuronal hyperexcitability by a presumably faster action potential repolarization 534

(Du et al., 2005). Additional studies suggested that depending on the distribution of the various β 535

subunits in the brain, this mutation can differently modulate K

channels contributing to the 536

pathophysiology of epilepsy and dyskinesia (Lee and Cui, 2009). As far as genes different from 537

KCNMA1, a polymorphism in KCNMB4, named rs398702, was also associated with mTLE in an 538

Irish cohort population (Cavalleri et al., 2007) but the study failed to be replicated (Manna et al., 539

2013), while a truncation mutation in KCNMB3 (p.Val256TyrfsTer4) affecting synaptic inhibition 540

and thereby increasing neuronal excitability and seizure susceptibility, was associated with 541

idiopathic generalized epilepsy (Hu et al., 2003; Lorenz et al., 2007).

542 543 544

CONCLUDING REMARKS 545

Epilepsy is one of the most common chronic and heterogeneous neurological disorders, affecting 1- 546

2% of the population, characterized by recurrent unprovoked seizures due to abnormal 547

synchronized electrical discharges within the CNS (Stenlein, 2004). Since ion channels mediate the 548

axonal conduction of action potentials and transduction through synaptic transmission, increasing 549

evidence suggests that any mutation-induced channel malfunction directly alter brain excitability 550

Provisional

12 and can induce epileptic seizures. Therefore, the discovery of genetic defects and, in particular, the 551

electrophysiological characterization of mutant ion channels in hereditary forms of epilepsy may 552

elucidate pathophysiological concepts of hyperexcitability in the CNS. This knowledge could 553

enable new therapeutic strategies by antagonizing the epilepsy-causing mechanisms using the 554

defective proteins as pharmacological targets. Given these considerations, we present an overview 555

of mutations in K

channels and their related accessory subunits underlying different human 556

epileptic phenotypes. Several families of K

channels have been involved in the pathogenesis of 557

epilepsy or other syndromes showing seizures as a clinical sign. For each channel family, the effect 558

of reported mutations is different: loss-of-function as well as gain-of-function could be observed.

559

The common effect of all mutations is to determine membrane iperexcitability, thus increasing the 560

susceptibility to seizures. Our review highlights the pleiotropic effects of some mutations in K

561

channels and the lack of a direct genotype-phenotype correlation. Interestingly, K

channels 562

dysfunctions seem to be mainly observed in epileptic patients with neurological comorbidities, such 563

as ASDs, intellectual disabilities or psychiatric features, in which they are associated with more 564

clinical severity. This observation could suggest to perform a mutation screening of K

channels in 565

patients showing intellectual disabilities.

566

In conclusion, the discovery of K

channels encoding genes that influence susceptibility and disease 567

progression will provide insight into the molecular events of epileptogenesis, improve molecular 568

diagnostic utility and identify novel therapeutic targets for treatment of human epilepsy.

569 570 571

Conflict of interest statement 572

The authors declare that they have no potential conflict of interests with the study.

573 . 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600

Provisional

13 REFERENCES

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